This invention relates to a process for producing recombinant human Cu,Zn-superoxide dismutase (to be referred to as "recombinant human SOD"). More specifically, it relates to a process for producing recombinant human SOD which comprises treating an apoenzyme of recombinant human superoxide dismutase with a copper salt in the presence of .beta.-mercaptoethanol.
Human SOD is a metalloenzyme having the action of scavenging superoxide (.O.sub.2.sup.-) in accordance with a disproportionation reaction of the following formula. ##STR1## Hence, human SOD is expected to be an effective therapeutic agent for tissue damage induced by superoxide generated from oxygen in vivo, for example inflammation, arthrosis deformans, chronic articular rheumatism, radiation damage, ultraviolet damage, retrolentri fibroplasia, cataract, side-effects of anticancer agents such as adriamycin, and injuries associated with reperfusion of blood to an ischemic part.
Methods have already been disclosed for the production of human SOD from human cells or tissues (see Japanese Laid-Open Patent Publications Nos. 141288/1982, 155991/1982 and 91881/1984). These methods, however, are not entirely suitable for industrial production because the raw materials are difficult to obtain in large quantities, and human placenta as a relatively easily available material has a low SOD content.
Much work has been done on the production of human SOD by the gene recombinant technology, and various methods have been disclosed to date (see Japanese Laid-Open Patent Publications Nos. 137286/1985, 111690/1986, 111693/1986 and 139390/1986). As stated in Japanese Laid-Open Patent Publication No. 111693/1986, recombinant human SOD accumulated as an intracellular product obtained by cultivating a microorganism transformed with a recombinant DNA having a human SOD structural gene downstream of an expression regulatory gene has quite the same amino acid sequence as natural human SOD. However, it does not contain a theoretical amount of a copper ion which is the active site of human SOD, and therefore has low enzyme activity.
J. C. Dinbar, B. Holmquist and J. T. Johansen reported (Biochemistry, 23, 4330-4335, 1984) that when a Cu-free apoenzyme of Cu,Zn-SOD is treated with a copper salt, a copper ion is taken into the apoenzyme to recover enzyme activity.
We therefore extensively studied the method of treating an apoenzyme of recombinant human SOD in a solution of a copper salt in order to produce recombinant human SOD containing a theoretical amount of a copper ion and having high enzyme activity. Consequently, we have succeeded in obtaining recombinant human SOD containing a theoretical amount of a copper ion and having high enzyme activity. However, this method gives rise to a new problem in that it gives recombinant human SODs having different electric charges and different molecular weights as shown by their electrophoresis.